The bacterial flagellum is a rotative filamentous appendage which is anchored in the bacterial membranes and allows the bacterium to move. Besides its main locomotive…
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The bacterial flagellum is a rotative filamentous appendage which is anchored in the bacterial membranes and allows the bacterium to move. Besides its main locomotive function, the bacterial flagellum fulfills other functions such as adhesion and secretion. The filament, which comprises most of the flagellum structure, is formed by an assembly of proteins called flagellins. It is estimated that nearly 80% of the bacteria produce a flagellum. Brucella is a mammal pathogens bacterium that had long been designated as non-flagellated. However, recent discoveries have shown that all flagellar genes were present in its genome and were necessary to Brucella’s pathogenic ability. Several regulators that control the production of flagellar proteins have also been uncovered. However, no flagellum was viewed on Brucella until today.
In this work, we showed for the first time through the optical and electron microscopy that B. melitensis produces transitionally a polar sheathed flagellum. On the other hand, we also investigated the regulation of flagellar system. Firstly, we studied the role of sigma factor RpoE1 on flagellar genes. The rpoE1 mutant overexpresses flagellar genes and produces a longer flagellum. Secondly, we showed that the production of flagellin was controlled by specific regulators named FlbT and FlaF. FlbT is a flagellin activator while FlaF is a flagellin inhibitor. In addition, we showed that the function of FlbT appears to be conserved among the Rizhobiales. We also showed that B. melitensis is more virulent in the absence of flagellin, which indicates a specific role of flagellin. The function of the flagellin of Brucella remains to be investigated.
Finally, a preliminary in silico analysis of the genome of Brucella has enabled us to target other potential regulators of the flagellar system. The data gathered during this work have allowed us to draw a hierarchical regulatory cascade of flagellar genes of Brucella in 3 classes.

▼ Mycobacterium avium subsp. paratuberculosis, the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools and vaccines. The first generation vaccines, composed of whole mycobacteria (killed or live-attenuated) in oily adjuvant confer a partial protection, by delaying the excretion of the mycobacteria in the faeces and by reducing the number of animals progressing to the clinical phase. However, they interfere with the diagnostic test of bovine tuberculosis making their use in cattle problematic. It is important to develop a new generation of vaccines which will protect better from the infection and which will not interfere with the screening tests of tuberculosis and paratuberculosis.
M. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. This particularity hampers infection, immunity and vaccination studies. In order to facilitate the screening of new vaccine candidates we have developed a luminescent M. paratuberculosis expressing luxAB genes of Vibrio harveyi and we have described its use for vaccine testing in an experimental mouse model, replacing fastidious and costly enumeration of CFU on agar by easy and rapid luminometry. Using this luminescent isolate, we have re-evaluated the effect of murine Slc11a1 (formerly called Nramp1) polymorphism on susceptibility to M. paratuberculosis. A series of inbred mouse strains were infected intravenously with luminescent M. paratuberculosis S-23 and monitored for bacterial replication in spleen, liver, and lungs for 12 weeks. The results confirm that, as for M. avium, innate resistance to infection is genetically controlled by Slc11a1. In BALB/c H-2d, congenic BALB.B10 H-2b (BALB/c background; H-2b), C57BL/6 H-2b, and beige C57BL/6bg/bg mice (all four homozygous for the susceptible Slc11a1s allele) bacterial numbers in spleen and liver remained unchanged during the first 4 weeks of infection. In DBA/2 and congenic BALB/c.DBA/2 (C.D2) mice (both homozygous for the resistant Slc11a1r allele) and in (C57BL/6 x DBA/2)F1 mice (heterozygous Slc11a1s/r), bacterial numbers decreased more than 10-fold during the first 4 weeks of infection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver.
Whereas bacterial numbers in the liver gradually decreased more than 100-fold in C57BL/6 mice between week 4 and week 12, bacterial numbers were stable in livers from BALB/c and beige C57BL/6bg/bg mice during this period. Mycobacterium-specific gamma interferon responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice.
In our screening of new vaccine candidates, we evaluated the immunogenicity and the protective potential of ten M.…
Advisors/Committee Members: FUNDP - SBIO_URBM (unité de recherche en biologie moléculaire), Letesson, Jean-Jacques, Muylkens, Benoit, Wattiez, Ruddy, Herman, Lieve, Huygen, Kris.

The PrlS/PrlR two component signal transduction system (TCS) is highly conserved in sequence and genomic organization in Brucella species, but its function remains completely unknown.…
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The PrlS/PrlR two component signal transduction system (TCS) is highly conserved in sequence and genomic organization in Brucella species, but its function remains completely unknown. TCS regulate genetic expression in response to external or internal environmental signals. In this work PrlS/PrlR mutants were characterized with results suggesting that wt B. melitensis produces aggregates at high osmolarity, contrary to the mutants of the bacterial pathogen Brucella melitensis. Interestingly it could be observed that the matrix of this aggregates contain EPS and that PrlR regulates positively the synthesis and production of EPS. We further investigated the role of prlR on membrane structures such as the flagellum and outer membrane proteins, by studying the regulation of the pfliF promoter in the mutants. Our results suggest that prlR controls both the flagellar system and the regulation of OMPs. B. melitensis mutated for prlR is less virulent compared to the wt suggesting that PrlR may play an important role in virulence. Preliminary transcriptomic comparative analysis of the mRNA profiles of mutant and wild type prlR strains suggests that PrlR regulates approximately 6.41 % of the B. melitensis genome.

DNA microarrays allow to study the expression profile of the whole genome of an organism. This technology is quite expensive, and the number of tested…
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DNA microarrays allow to study the expression profile of the whole genome of an organism. This technology is quite expensive, and the number of tested samples is often limited (at most 5 replicates). In those conditions, statistical tests are associated to bad performances. Various methods have been developed to optimize differential expression analysis. We describe a set of methods, to provide a comprehensive view of various approaches, of their main advantages and limitations.
Our first objective is to guide the analysis of differential expression, at the gene-level, by using biological or empirical informations. To improve the performances of statistical tests, we propose to share information across genes, gathered using an appropriate criteria. The window t-test has been developed following this strategy, to use the empirical relationship between variability and mean expression level. The window t-test only depends on the number of replicates, that defines the number of probesets used to compute variance estimates. Evaluation of methods reveals that the window t-test performs similarly to or better than the best methods [1].
Many biological informations can be used to define gene-sets (metabolic pathway, chromosomal location...). Current methods for gene-set analysis of differential expression are developed to test several hypothesis. We generalize gene-set analysis to answer the main Q0 question : « Does the individual expression values of the gene-set members differ between two condition ? ». We developed FAERI to answer to this question, by considering 3 criteria : the correlation between genes, the expression level, and the direction of the response (under- or over-expression). FAERI is a modified ANOVA-2 procedure, starting with a two-step reduction of expression data (Z-standardization, directional reduction). ANOVA-2 is shown to be the best-performing method when analyzing uni-directional gene-sets (all members are either activated, or repressed). FAERI reveals to be the most appropriate method for all tested gene-set types.
We developed PEGASE to perform differential expression analysis both at the gene and gene-set level. Consensus evaluation from several methods is included, to provide users with good results, even if the choice of an optimal method is not easy. Several methods are implemented in PEGASE, both at the gene and gene-set level, and performance evaluation can be run based on biological or empirical knowledge. PEGASE is also used as a back-end by PHOENIX, an online tool for microarray data analysis [2].
1. Berger F., De Hertogh B., Pierre M., Gaigneaux A. & Depiereux E. The "Window t-test": a simple and powerful approach to detect differentially expressed genes in microarray datasets. Cent. Eur. J. Biol., 2008, 3, 327-344.
2. Berger F., De Hertogh B., Bareke E., Pierre M., Gaigneaux A. & Depiereux E. PHOENIX: a web-interface for (re)analyses of microarray data. Cent. Eur. J. Biol., 2009, 4(4) : 603 : 618.

► Brucellosis is a bacterial zoonose caused by organisms belonging to the genus Brucella. These bacteria are facultative intracellular pathogens that cause abortion in domestic (cattle,…
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▼ Brucellosis is a bacterial zoonose caused by organisms belonging to the genus Brucella. These bacteria are facultative intracellular pathogens that cause abortion in domestic (cattle, goats, sheeps, etc.) and wild (deers, bisons, etc.) animals and a febrile chronic illness in humans. The disease exists worldwide and continues to have a great health significance and economic importance in developing countries. Despite past and current efforts to eradicate brucellosis by vaccination and culling within cattle and herds, as many as 500 000 new human cases are reported annually worldwide. Vaccines against brucellosis were initially developed on an empirical basis. The current studies are moving towards a more rational design but are hindered by an incomplete knowledge of the in vivo life style of Brucella and the immune mechanisms involved in the establishment of a protective memory. Despite progresses, several fields of host adaptive immunity remain highly controversial such as the implication of B cells, CD4+ and CD8+ T cells. While it is commonly accepted that Brucella infection induces a Th1 immune response characterized by the production of IFN-γ that activates bactericidal mechanisms of macrophages, little has been described with regards to a potential implication of the Th2 response or the recently described Th17 response.
Objectives of this thesis were therefore to determinate the implication of lymphocyte subsets and signaling pathways after primary infection with Brucella melitensis and characterize their roles in the development of a protective secondary immune response in the murine experimental model.
In the first part of this study we clearly confirm the central role of MHC-II-dependent antigen presentation to CD4+ T cells and the IFN-γ-mediated Th1 response in the control of B. melitensis primary infection. We also report that the absence of B cells, MHC-I-dependent antigen presentation, Th2 and Th17 responses appears to have no important positive or negative impact on the course of infection.
In the second part of this work, we show that Brucella is able to persist several weeks in the blood of infected mice. Surprisingly, we found that bacteria are initially localized extracellularly and then infect erythrocytes where they are already detectable after 24h.
Finally, the last part demonstrates that humoral immunity and CD4+ Th1 cells are both necessary and complementary for a sterilizing immune response upon a secondary infection with B. melitensis. Circulating specific antibodies and IFN-γ production by CD4+ T cells activated at the site of infection after the re-call infection appear as key immunological markers of protection in the murine experimental model of Brucella infection.
In conclusion, this work improves our understanding of the nature of murine immune response developed following B. melitensis infection and tries to provide correlates of protection that could help to define rational strategies for designing new vaccines against brucellosis. This study also reveals for the first time that…
Advisors/Committee Members: FUNDP - SBIO_URBM (unité de recherche en biologie moléculaire), FUNDP - Ecole doctorale en sciences, Letesson, Jean-Jacques, Muraille, Eric, Michiels, Carine, Leo, Oberdan, Goddeeris, Bruno, Michiels, Carine, Baldwin, Cynthia.

Vitry, M. (2013). On the components of adaptive immune response involved in the control of a primary and secondary infection by Brucella melitensis. (Thesis). DIAL (Belgium). Retrieved from http://hdl.handle.net/2078.2/129079

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Vitry, Marie-Alice. “On the components of adaptive immune response involved in the control of a primary and secondary infection by Brucella melitensis.” 2013. Thesis, DIAL (Belgium). Accessed May 25, 2019.
http://hdl.handle.net/2078.2/129079.

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Vitry, Marie-Alice. “On the components of adaptive immune response involved in the control of a primary and secondary infection by Brucella melitensis.” 2013. Web. 25 May 2019.

Vancouver:

Vitry M. On the components of adaptive immune response involved in the control of a primary and secondary infection by Brucella melitensis. [Internet] [Thesis]. DIAL (Belgium); 2013. [cited 2019 May 25].
Available from: http://hdl.handle.net/2078.2/129079.

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Vitry M. On the components of adaptive immune response involved in the control of a primary and secondary infection by Brucella melitensis. [Thesis]. DIAL (Belgium); 2013. Available from: http://hdl.handle.net/2078.2/129079

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

Adaptation and stress response rely on the coordination of several molecular actors involved in stimuli perception, signal integration or the establishment of the adaptative response.…
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Adaptation and stress response rely on the coordination of several molecular actors involved in stimuli perception, signal integration or the establishment of the adaptative response. In pathogenic bacteria, the regulatory mechanisms are essential to ensure an successful infectious process. Under certain conditons, the response of the bacteria leads to biofilm formation which are often associated with resistance mechanisms.
During its lifetime, B. melitensis might be facing changing environmental conditions sometimes harmful. Mutants altered in Quorum Sensing system form bacterial clusters structurally similar to biofilms. The study of these aggregates revealed that they were predominantly composed of exopolysaccharide (EPS). However, the conditions under which this EPS is produced by the wild type bacterium, its role and the genes encoding the enzymes responsible for its biosynthesis remained unknown. In this study, we demonstrated the formation of aggregates in response to salt stress by wild type Brucella spp. We showed that the formation of these bacterial clumps depends on the presence of a phosphorelay PrlS/PrlR and of the ionic strength of the growth medium. Our data, however, challenged the exitence of a production of an exopolysaccharide by Brucella spp. Finally, we studied the role of two regulators, the system PrlS/PrlR and the transcriptional regulator MucR in the formation of aggregates in response to salt stress and in the virulence of B. melitensis. Our data indicate that the system PrlS/PrlR, required for persistent infection in vivo, is involved in the response to salt stress and regulate actors probably required for aggregation. Moreover, MucR is a key regulator of stress response and is involved in intracellular survival of the bacteria. We showed that it acts as a repressor of flagellar gene expression and is required for growth of B. melitensis in hypersaline conditions. If MucR involvement in the regulation of EPS remains to be shown in B. melitensis, our data shows its role in the regulation of the structure of another major component of the bacterial surface, the lipopolysaccharide.

► Modifications of transfer RNA (tRNA) are widespread and numerous but their function is not well understood. Particularly, three tRNAs (tRNALysAAA, tRNAGluGAA and tRNAGlnCAA) show universal…
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▼ Modifications of transfer RNA (tRNA) are widespread and numerous but their function is not well understood. Particularly, three tRNAs (tRNALysAAA, tRNAGluGAA and tRNAGlnCAA) show universal modifications of the second and fifth carbons of the wobble base. The «Elongator» complex (Elp1-6) is proposed to modify carbon five while the Ctu1-Ctu2 complex is responsible for the thiolation of carbon two. In both cases, the modification is thought to be required to optimize the codon-anticodon interaction. In the fission yeast, the deletion of either complexes results in thermosensitivity associated with growth defects on specific compounds as well as cell cycle and cytokinesis defects. A key step in understanding how these phenotypes result from a tRNA modification defect is to compare the proteome from wild type and mutants.
We determined the proteome-wide relative expression level of each S. pombe protein by combining an integrated ORFeome to reverse protein arrays. We identified subsets of proteins whose expression level was altered in the strains lacking either modification. The groups identified can be linked to the phenotypes observed. As a detailed example, the subset of proteins involved in the « mitotic cell cycle transition from G2 to M » has been thoroughly studied. Several genes coding for proteins belonging to this subset show genetic interaction with ctu1 or elp3. One of the more interesting of these genes is cdr2, coding for a protein kinase negatively regulating Wee1 to allow the entry into mitosis. The cell cycle phenotypes associated with the absence of tRNA modifications can be explained by the strong decrease in Cdr2 level observed in the corresponding mutants. Moreover, the ctu1 and elp3 mutant phenotypes mimic most of the reported
cdr2 mutant phenotypes. Yet, these phenotypes are aggravated in the triple ctu1 elp3 cdr2 mutant, suggesting that Cdr2 is not the only protein responsible for the phenotypes observed in absence of modification. Why is the translation of the proteins in these subsets – and particularly Cdr2 – hyper sensitive to the absence of mcm5s2U tRNA modification? The answer is found in the codon content of the corresponding genes. Indeed, all the sensitive groups are enriched in AAA codons (by opposition to AAG), which is read by tRNALysUUU, one of the three mcm5s2U modified tRNAs. This link between codon content and sensitivity to the absence of tRNA modification has been demonstrated experimentally via the study of Cdr2. We suppressed the translation defect of Cdr2 by overproducing tRNALysUUU from a plasmid, or by replacing every AAA lysine codon by a AAG-lysine (read by the naturally unmodified tRNALysCUU).
Taken all together, these experiments show that the translation of groups of genes enriched in AAA codon is less efficient in the absence of mcm5s2U modification. How is the selection pressure leading to an enrichment of these genes in AAA codons exerted? Why enriching a gene with the lysine AAA codon that is read with low efficiency? These issues are part of the remaining…
Advisors/Committee Members: FUNDP - SBIO_URBM (unité de recherche en biologie moléculaire), FUNDP - Ecole doctorale en sciences, Damien, Hermand, Jean-Jacques, Letesson, Anders, Bystrom, Alain, Chariot, Annabelle, Decottigines, Pasty, Renard.

Metastases are the final stage of cancer development and are responsible for many of the deaths from cancer. Recent studies have described the signaling pathways…
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Metastases are the final stage of cancer development and are responsible for many of the deaths from cancer. Recent studies have described the signaling pathways associated with metastatic phenotype. However, the actual involvement of these pathways in the initiation of metastasis is not always well understood.
Another key feature of tumors is the presence of hypoxic areas caused by a lack of oxygen in the center. Hypoxia leads to the expression of genes promoting the formation of metastases and the repression of genes that prevent their development.
Many studies of metastasis and / or the response to hypoxia have used DNA microarrays. However, they rarely have fully exploited this technique. As the data generated were made public, this work offers a re-analysis of these data sets to extract new information on the metastatic phenotype induced by hypoxia in several cancer cell lines, as well as validations of the hypotheses generated in silico.
Three complementary approaches were developed that allowed the selection of 165 genes of interest. Among these 165 genes, 91 have already been described in the literature as being involved in cancer. Among which 41 are known to regulate the metastatic potential. Twenty genes were also described to be involved in the response to hypoxia. Finally, these 165 genes can be classified into 42 different signaling pathways, among which 12 are directly linked to cancer, while 5 others are related to proliferation and cell motility. 1000 negative controls consisting of 165 randomly selected genes were unable to provide such results. Surprisingly, 5 signaling pathways, in which are involved the 165 genes of interest are related to pathogen recognition and to phagocytosis.
The expression profiles of 9 genes involved in one of the pathways of phagocytosis and one of the pathways of pathogen recognition were validated by real time RT-PCR. Migration tests were performed for three of these genes and two of them (PAK1 and CFL2) clearly show an involvement in cell migration providing new targets in the fight against this disease.

The objective of this project is to reduce the background noise which limits the interpretation of experiments using microarrays, by integrating information from genomic databases…
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The objective of this project is to reduce the background noise which limits the interpretation of experiments using microarrays, by integrating information from genomic databases to statistical analysis. Statistical methods generate a high background noise, filtered today only downstream of the analysis, by analytical tools and bibliographic biological information. They are unable to filter false negatives according to statistical criteria.
These criteria are generally used independently of each other and independently for each gene. Their consideration is neither automatic nor weighted.
To filter the results of the analysis of microarrays, a promising way is to submit to the statistical analysis subsets of genes between which relations have been established in reference to bioinformatics databases. The aim of the project is to develop a relational database specifically dedicated to the analysis of these results, by crossins information from numerous available databases.
A request may be structured via an expert system and this information may be integrated at various levels of the decision-making statistical methodology to increase substantially its power.

Cyclin-dependent kinases (CDK) belong to a group of kinases involved in both cell cycle control and transcription regulation. To be fully active, CDKs require phosphorylation…
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Cyclin-dependent kinases (CDK) belong to a group of kinases involved in both cell cycle control and transcription regulation. To be fully active, CDKs require phosphorylation by a Cdk-activating kinase (CAK). The fission yeast Schizosaccharomyces pombe possesses the two types of CAK : trimeric Mcs6-Mcs2-Pmh1 complex and monomeric Csk1 protein. Mcs6 and Csk1 can both phosphorylate and activate Cdc2, the main cell cycle regulator, in vitro. However, strong genetic data indicate that only Mcs6 activates Cdc2 in vivo. Analysis of an analog-sensitive mutant of Mcs6 show that the sole inactivation of Mcs6 is necessary and sufficient to abolish Cdc2 phosphorylation in vivo. Our data also indicate that Csk1, contrary to the related kinase Cak1, is unable to precipitate and phosphorylate Cdc2 from a fission yeast lysate. Here we establish that Mcs6 is the genuine CAK in vivo of Cdc2 in S. pombe.